Photonic crystal sensors with intergrated fluid containment structure

ABSTRACT

Photonic crystal (PC) sensors, and sensor arrays and sensing systems incorporating PC sensors are described which have integrated fluid containment and/or fluid handling structures. Sensors and sensing systems of the present disclosure are capable of high throughput sensing of analytes in fluid samples, bulk refractive index detection, and label-free detection of a range of molecules, including biomolecules and therapeutic candidates. The present disclosure also provides a commercially attractive fabrication platform for making photonic crystal sensors and systems wherein an integrated fluid containment structure and a photonic crystal structure are fabricated in a single molding or imprinting processing step amendable to high throughput processing.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to the provisions of 35 U.S.C. §119 (e), this applicationclaims priority to U.S. Provisional Application Ser. No. 60/865,093filed Nov. 9, 2006, the content of which is incorporated by referenceherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, at least in part, with United Statesgovernmental support awarded by National Science Foundation under NSFDM1 03-28162. The United States Government has certain rights in thisinvention.

BACKGROUND OF DISCLOSURE

Photonic crystals, also commonly referred to as photonic bandgapstructures, are periodic dielectric or metallic structures exhibiting aspatially periodic variation in refractive index that forbidspropagation of certain frequencies of incident electromagneticradiation. The photonic band gap of a photonic crystal refers to therange of frequencies of electromagnetic radiation for which propagationthrough the structure is prevented. The photonic band gap phenomenon maybe conceptualized as complete reflection of incident electromagneticradiation having selected frequencies due to interaction with theperiodic structural domains of a photonic crystal. The spatialarrangement and refractive indices of these structural domains generatephotonic bands gaps that inhibit propagation of electromagneticradiation centered about a particular frequency. Background informationon photonic crystals include the following references: (1) Joanopouluset al., “Photonic Crystals Molding the Flow of Light”, PrincetonUniversity Press, 1995; (2) A. Birner, et al., “Silicon-Based PhotonicCrystals”, Advanced Materials, Volume 13, Issue 6, Pages 377-388; and(3) Steven G. Johnson, and John D. Joannopoulos, “Photonic Crystals: TheRoad from Theory to Practice”, Springer, 2002.

Photonic crystals provide an electromagnetic analog to electron-wavebehavior observed in crystals wherein electron-wave concepts, such asdispersion relations, Bloch wave functions, van Hove singularities andtunneling, having electromagnetic counterparts in photonic crystals. Insemiconductor crystals, for example, an electronic band gap of energystates for which electrons are forbidden results from a periodic atomiccrystalline structure. By analogy, in a photonic crystal, a photonicband gap of forbidden energies (or wavelengths/frequencies) ofelectromagnetic radiation results from a periodic structure of adielectric material where the periodicity is of a distance suitable tointeract with incident electromagnetic radiation.

Selection of the physical dimensions, refractive indices and spatialdistribution of periodic structural components (“surface grating”herein) of a photonic crystal provides an effective means of designing aphotonic crystal a photonic band gap with a selected frequencydistribution. If the periodicity and symmetry of the crystal and thedielectric constants of the materials used are chosen appropriately, thephotonic crystal will selectively couple energy at particularwavelengths, while excluding others. One-dimensional, two-dimensionaland three-dimensional photonic crystals have been fabricated providingcomplete or at least partial photonic band having selected frequencydistributions gaps in one or more directions. Photonic crystals havealso been fabricated having selected local disruptions (e.g., missing ordifferently-shaped portions of the structural domains of periodic array)in their periodic structure, thereby generating defect or cavity modeswith frequencies within a forbidden bandgap of the crystal. Photoniccrystals having specific defects are of particular interest because theyprovide optical properties useful for controlling and manipulatingelectromagnetic radiation, such as the ability to provide opticalconfinement and/or wave guiding with very little, or substantially no,radiative losses. U.S. Pat. No. 6,990,259 to Cunningham describes a“defect” biosensor in greater detail. The content of the '259 patent isincorporated by reference herein.

As diffraction and optical interference processes give rise to thephotonic band gap phenomenon, the periodicity of photonic crystalstructures is typically on the order of the wavelength of incidentelectromagnetic radiation. Accordingly, photonic crystals forcontrolling and manipulating visible and ultraviolet electromagneticradiation typically comprise dielectric or metallic structures withperiodic structural domains having submicron physical dimensions on theorder of 100's of nanometers. A number of fabrication pathways formaking periodic structures having these physical dimensions have beendeveloped over the last decade, including micromachining andnanomachining techniques (e.g., lithographic patterning and dry/wetetching, electrochemical processing etc.), colloidal self assembly,replica molding, layer by-layer assembly and interference lithography.Advances in these fabrication techniques have enabled fabrication ofone-dimensional, two-dimensional and three-dimensional photonic crystalsfrom a range of materials including dielectric crystals, metals,polymers and colloidal materials.

The applications of photonic crystal sensors are numerous, includingintegration with lasers to inhibit or enhance spontaneous emission,waveguide angle steering devices, and as narrowband optical filters. Aphotonic crystal structure geometry can be designed to concentrate lightinto extremely small volumes and to obtain very high localelectromagnetic field intensities.

In order to adapt a photonic crystal device to perform as a biosensor,some portion of the structure must be in contact with a test sample. Byattaching biomolecules or cells to the portion of the photonic crystalwhere the locally confined electromagnetic field intensity is greatest,the resonant coupling of light into the crystal is modified, so thereflected/transmitted output is tuned. The highly confinedelectromagnetic field within a photonic crystal structure provides highsensitivity and a high degree of spatial resolution consistent withtheir use in imaging applications, much like fluorescent imagingscanners.

For example, photonic crystals with subwavelength periodic gratingstructures have been developed to reflect only a very narrow band ofwavelengths when illuminated with white light. To create a biosensor, aphotonic crystal may be optimized to provide an extremely narrowresonant mode whose wavelength is particularly sensitive to modulations(i.e., shifts) induced by the deposition of biochemical material on itssurface. In typical practice, a photonic crystal sensor consists of alow refractive index plastic material with a periodic surface structurethat is coated with a thin layer of high refractive index dielectricmaterial. The sensor is measured by illuminating the surface with whitelight, and collecting the reflected light with a non-contact opticalfiber probe, where several parallel probes can be used to independentlymeasure shifts in the peak wavelength of reflected light (“PWV”) atdifferent locations on the sensor. The biosensor design enables a simplemanufacturing process to produce sensor sheets in continuous rolls ofplastic film that are hundreds of meters in length. The massmanufacturing of a biosensor structure that is measurable in anon-contact mode over large areas enables the sensor to be incorporatedinto single-use disposable consumable items such as 96, 384, and1536-well standard microplates, thereby making the sensor compatiblewith standard fluid handling infrastructure employed in mostlaboratories. In these cases, the photonic crystal is manufactured inseparate manufacturing operation, and then, in a second step, glued orotherwise adhered to a bottomless microplate. The wells of themicroplates provide a reservoir by which a fluid sample can beintroduced onto the photonic crystal surface.

The sensor operates by measuring changes (shifts) in the wavelength ofreflected light (“PWV”) as biochemical binding events take place on thesurface. For example, when a protein is immobilized on the sensorsurface, an increase in the reflected wavelength is measured when acomplementary binding protein is exposed to the sensor. Using low-costcomponents, the readout instrument is able to resolve protein masschanges on the surface with resolution less than 1 pg/mm². While thislevel of resolution is sufficient for measuring small-moleculeinteractions with immobilized proteins, the dynamic range of the sensoris large enough to also measure larger biochemical entities includinglive cells, cell membranes, viruses, and bacteria. A sensor measurementrequires about 20 milliseconds, so large numbers of interactions can bemeasured in parallel, and kinetic information can be gathered. Thereflected wavelength of the sensor can be measured either in “singlepoint mode” (such as for measuring a single interaction within amicroplate), or an imaging system can be used to generate an image of asensor surface with <9 μm resolution. The “imaging mode” has been usedfor applications that increase the overall resolution and throughput ofthe system such as label-free microarrays, imaging plate reading,self-referencing microplates, and multiplexed spots/well.

Given substantial advances in their fabrication and their unique opticalproperties, photonic crystal-based sensors have been recently developedfor a range of biosensing applications. To operate as a biosensor, aphotonic crystal is provided in a configuration such that its activearea is exposed to a fluid containing analytes for detection. Thepresence of analyte proximate to the photonic crystal sensor modulatesthe resonant coupling of light into the crystal, thereby resulting in ameasurable change in the wavelength distribution of electromagneticradiation transmitted, scattered or reflected by the crystal resultingfrom changes in the photonic band gap of the crystal. The highlylocalized nature of the confined electromagnetic field generated by thecrystal ensures that that detection via photonic crystal based sensorsis restricted to a probe region proximate to (e.g., 100-400 nanometers)the active area of the sensor. In typical sensing applications, a readout system is used wherein polarized electromagnetic radiation having aselected wavelength distribution is provided to the photonic crystal andsubsequently reflected or transmitted electromagnetic radiation isfrequency analyzed by an appropriate photodetector, such as aspectrometer in combination with an appropriate detector. By observingand/or quantifying the change in wavelength distribution resulting frominteraction of the fluid and the photonic crystal, analytes in the proberegion are detected and/or analyzed.

Biosensors incorporating photonic crystal structures are described inthe following references which are hereby incorporate by reference: U.S.Pat. Nos. 7,118,710, 7,094,595, 7,023,544, and 6,990,259; andCunningham, B. T., P. Li, B. Lin and J. Pepper, Colorimetric ResonantReflection as a Direct Biochemical Assay Technique, Sensor and ActuatorsB, 2002, 81, pgs 316-328; and Cunningham, B. T. J. Qiu, P. Li, J. Pepperand B. Hugh, A Plastic Calorimetric Resonant Optical Biosensor forMultiparallel Detection of Label 10 Free Biochemical Interactions,Sensors and Actuators B, 2002, 85, pgs 219-226.

Advantages provided by photonic crystals for biosensing include theability to detect and characterize a wide range of materials, includingpeptides, proteins, oligonucleotides, cells, bacteria and virusparticles, without the use of labels, such as fluorescent labels andradioligands, or secondary reporter systems. Direct detection providedby photonic crystal sensing enhances easy of implementation of thesetechniques by eliminating labor intensive processing required tosynthetically link and/or read out a label or reporter system. Thisbeneficial aspect of photonic crystal-based sensing also eliminates asignificant source of experimental uncertainty arising from theinfluence of a label or reporter system on molecular conformation,reactivity, bioactivity and/or kinetics; and eliminates problems arisingfrom liquid phase fluorescence quenching processes. Photonic crystalbased sensors are also compatible with functionalization, for example byincorporation of biomolecules and/or candidate therapeutic moleculesbound to the surface of the active area of the photonic crystalstructure; a capability which is particularly attractive for selectivelydetecting specific target molecules for screening and biosensingapplications. Other benefits provided by photonic crystal approaches tobiosensing include: (i) good sensitivity and image resolution; (ii)compatibility with relatively straightforward optical readout systems,(iii) and the ability to provide highly localized detection useful formultichannel systems having a high area density sensors are emerging asa major tool for selective biochemical detection and analysis in diversefields including genomics, proteomics, pharmaceutical screening andbiomedical diagnostics.

In current practice, photonic crystal biosensors and the associatedlarger-scale fluid containment features (such as wells or channels) aretypically fabricated separately and subsequently integrated viaalignment and bonding processes. Given the submicron scale of featuresof the photonic crystal and micron or larger scale physical dimensionsof the fluid containment structures, alignment and bonding steps inphotonic crystal-based sensors present significant practical challenges,and thus add to the overall cost and complexity of fabrication of thesedevices. First, the components of photonic crystal biosensors areoptimally aligned such that the maximum extent of active area of thephotonic crystal is exposed to fluid held in the fluid containmentstructure. Second, bonding and alignment must effectively prevent liquidfrom exiting a given fluid containment structure and spreading to one ormore adjacent fluid containment structures in a multichannel sensorconfiguration. This requirement is necessary to avoid sensinginterferences arising from cross talk between adjacent photonic crystalsensors. Third, the force applied to the photonic crystal structureduring alignment and bond must be sufficiently low so as not to damagethe nanoscale periodic features of the crystal. Damage to such featurescan introduce unwanted defect structures to the photonic crystal thatcan strongly influence sensing capabilities and readout of the device.

SUMMARY

This disclosure is premised on the inventors' insight that photoniccrystal sensors are capable of integration in a monolithic device havingfluid containment structures such as wells or flow channels, includingarrays of wells and associated fluid flow channels. The sensors of thepresent disclosure have a great potential for implementation inmicrofluidic lab-on-a chip (LOC) devices, micro-total-analysis systems(μTAS) and biosensor—embedded microarray systems.

In these applications of the present disclosure, fluid containmentstructures, such as wells or fluid flow channels, are integrated withthe sensor directly resulting in a monolithic, integral structure. Thefluid containment structures can be designed to effectively convey thesample to the active area (periodic surface grating) of a photoniccrystal. In some applications, the fluid containment structures furtherfunction in multichannel biosensor configurations to provide a fluidsample to a selected photonic crystal in a manner preventing the fluidsample spreading between adjacent sensors on the same substrate. Flowcells, such as microfluidic channels, are commonly used to provide ameans for conveying a fluid sample through a narrow channel from asample reservoir to the photonic crystal structure for analysis. Typicalflow cell configurations employ an etched trench having an attachedcover plate. These fluid containment and delivery systems must beprecisely aligned to and effectively sealed against the photonic crystalactive area so as to prevent leakage of sample. In other embodiments,the fluid containment structure is static, such as a cuvette aligned andbonded over the photonic crystal active area. In these embodiments, thephotonic crystal sensor is provided as an internal surface of thecuvette. In multi-array configurations, for example, a large number ofcuvettes each having an individually-addressed, independent photoniccrystal sensors are provided in a microplate format, such as a 96, 384or 1536 microarray format.

In another aspect, a biosensor is described having a integral structurehaving an inlet port, a plurality of sample wells connected to the inletport, and a plurality of flow channels connecting the inlet port to thesample wells, and a plurality of photonic crystal sensors. The photoniccrystal sensors are positioned in a flow channel connecting the inletport to the sample wells. In one specific embodiment, each of the samplewells also includes a photonic crystal sensor.

The physical dimensions and shapes of the fluid containment and/orhandling structures can take a variety of forms. Some forms are usefulfor constraining, transporting or otherwise providing a fluid sample tothe photonic crystal sensor such that analytes in the sample can beeffectively detected and/or analyzed. Integrated fluid containmentand/or fluid handling structures of the present disclosure can alsoinclude active fluidic structures in the form of flow channels where thesample moves over the photonic crystal, such as microfluidic andnanofluidic flow channels. In other embodiments, the fluid containmentstructures are passive, and may take the form of cuvettes, wells andmicrowell arrays.

Embodiments of the present disclosure include fluid containmentstructures which are oriented in a substantially aligned configuration.For example, in an embodiment in which the fluid containment structuresinclude a multitude of channels each having a photonic crystal formed atthe bottom of the channel, the photonic crystals in the various channelsare aligned with each other, i.e., in a straight line. As such,measurements of all the photonic crystal sensors can be madesimultaneously in a line-scanning type imaging spectrometer detectionapparatus. The alignment is deterministically selected and controlledduring fabrication. The resulting biosensor provides good imageresolution, high sensitivities and detection efficiencies.

The biosensors and associated detection instruments of the presentdisclosure are capable of high throughput sensing of analytes in fluidsamples, bulk refractive index detection, and label-free detection of arange of molecules, including biomolecules and therapeutic candidates.The biosensors and associated detection instruments also provide imagingfunctionality wherein a spatial profile of the active area of a photoniccrystal sensor or array of photonic crystal sensors is characterizedwith good resolution and sensitivity. This functionality is particularlyuseful, for example, for providing imaging assays within a fluidicchannel or for reading out a plurality of microwells provided in amicroarray configuration.

The present disclosure also features commercially attractive fabricationmethods for making photonic crystal sensors, sensor arrays and systemswith integrated fluid containment structures. The fabrication methods ofthe present disclosure are capable of cost effective and high throughputimplementation for the manufacture of photonic crystal sensors,including polymer-based photonic crystal sensors. Some methods of thisaspect of the present disclosure use a processing strategy wherein anintegrated fluid containment structure and a photonic crystal structureare fabricated simultaneously via single step integration, amendable tohigh throughput processing. Useful processing methods of this aspect ofthe present disclosure include the use of replica molding and imprintlithography techniques. The methods enable automatic, high precisionalignment of both the photonic crystal sensors and the fluid containmentstructures so as to ensure high performance device functionality. Thepresent fabrication methods are particularly well suited for makingphotonic crystal sensors comprising polymer materials, includingmechanically flexible polymer based photonic crystal sensors andsystems, and making arrays of photonic crystal sensors covering largeareas, and optionally, provided in a dense area configurations.

In the context of this description, the expression “monolithicstructure” refers to a unitary structure having a plurality ofintegrally connected structural elements. An exemplary monolithicstructure in some embodiments comprises a plurality of integratedstructural elements comprising a structurally continuous material,including a structurally continuous composite (multilayered) material.In some embodiments, the monolithic structure of sensors of the presentdisclosure comprise a single, continuous polymer structure wherein thesurface grating structure of a photonic crystal forms part of the oneinternal surface of a fluid containment structure, e.g., the bottomsurface of a sample well or fluid flow channel. In some embodiments, themonolithic structure comprising the integrated fluid containment andphotonic crystal structures is a mechanically flexible monolithicpolymer structure. In other embodiments the monolithic structure isrigid. Embodiments of the present disclosure having such amulti-component monolithic structure are useful for providing a sensorin which the periodic surface grating area of the photonic crystal issubstantially aligned within a fluid containment structure, capable ofproviding efficient and sensitive detection and characterization ofanalytes. Further, such multi-component monolithic structures are usefulfor providing photonic crystal sensors that are not susceptible toproblems associated with fluid sample leaking out of the fluidcontainment structure of the sensor.

In an embodiment, the monolithic structure comprising the fluidcontainment structure and the photonic crystal structures has discretestructural domains, one corresponding to a fluid containment structureand the other corresponding to the periodic grating structure of thephotonic crystal sensor. The discrete structural domains may havesubstantially different physical dimensions, for example physicaldimensions that differ by at least one order of magnitude, and in someembodiments physical dimensions that differ by at least two orders ofmagnitude. For example, the photonic crystal surface grating structuremay be nano-sized features, whereas the cavity of the fluid containmentstructure (flow channel or well) may be a micro-sized feature. Thephysical dimensions and shapes of cavities of fluid containmentstructures of the present disclosure can vary significantly fordifferent sensing applications. Several possibilities include fluid flowchannels, cuvettes, and microwell and microarray configurations. Inrepresentative embodiments, the periodic grating is of asub-illumination wavelength size, for example having physical dimensionsselected over the range of about 20 nanometers to about 500 nanometers,whereas the fluid containment structures, such as cavities, channels andrecessed or grating structures, are in the range of about 10 microns toabout 1000 microns.

In an embodiment of this aspect of the present disclosure, the gratingstructure of the photonic crystal is provided on a bottom or internalsurface of the cavity of fluid containment structure, and in aconfiguration wherein the grating structures extend from one side of thecavity to the other side. For example, sensors of the present disclosureinclude configurations wherein the fluid containment structure is afluid flow channel having a surface grating structure extending from oneside of the channel to the opposite side. The periodic surface gratingcan take the form of a one dimensional spatially periodic configurationsuch as a parallel array of alternating high and low portions. Otherperiodic structures are possible including two-dimensional gratings(arrays of posts or holes) or two-level, two dimensional periodicstructures.

In an embodiment, the photonic crystal structure provided on theinternal surface of the fluid containment structure comprises adielectric and/or semiconductor structure having a spatial distributionof refractive indices that varies periodically in at least twodimensions. Sensors of this embodiment of the present disclosure, forexample, may comprise a photonic crystal structure having atwo-dimensional periodic array of alternating high refractive indexelements and low refractive index elements.

In one sensor configuration, high refractive index elements, such asthin dielectric and/or semiconductor films, are disposed on top surfacesof at least a portion of the periodic surface grating and on the bottomsurfaces of the grating. Thin films providing high refractive indexelements useful in the present disclosure have thicknesses selected overthe range of about 20 nanometers to about 500 nanometers and include,but are not limited to, TiO₂ films, silicon nitride, tantalum oxide,zinc sulfide, and hafnium oxide.

In the context of this description, “high refractive index elements”have a refractive index higher than “low refractive index elements”, forexample a refractive index at least 1.2 times larger than the lowrefractive index elements in some embodiments. In some sensors, thecombination of high refractive index thin films provided on top of lowrefractive index grating structures (and optionally on side surface ofgrating structures) results in a photonic crystal structure having aspatial distribution of refractive indices that varies periodically intwo dimensions. Sensors of the present disclosure include, additionally,photonic crystal structures having grating structures provided in aspatially periodic configuration that includes at least one defect sitein a one-, two-, or three dimensional array, such as a missing relieffeature(s), extra relief feature(s) or relief feature(s) havingdifferent physical dimensions. Sensors of the present disclosure canalso include photonic crystal structures provided on the internalsurface of the fluid containment structure comprising athree-dimensional periodic array of alternating high refractive indexelements and low refractive index elements.

Sensors of the present disclosure may have a wide variety of integratedfluid containment structures, including active fluidic delivery andhandling systems, passive fluid reservoirs and all combinations andarrays and systems thereof. In an embodiment, the cavity of the fluidcontainment structure is a fluidic channel, such as a microfluidic ornanofluidic channel. Fluidic channels useful as fluid containmentstructures of the present disclosure are optionally a component of anactive fluidic system having pumps, valves, reservoirs and/or fluidicchannel networks. In an embodiment, the cavity of the fluid containmentstructure is a static reservoir, such as a cuvette, microwell,microcuvette and microreservoir. Sensors of this aspect of the presentdisclosure may be provided in an array format wherein a plurality offluid containment structures comprising microwells are provided in amicroarray format, wherein each microwell has a photonic crystalstructure provided on an internal surface.

In some embodiments of this disclosure, the sensor further includes acover layer positioned to enclose and/or seal the cavity of the fluidcontainment structure. Cover layers of this embodiment may optionally bebound to the fluid containment structure so as to prevent leakage andfacilitate handling of a fluid sample, for example using an adhesivelayer positioned between the cover layer and the fluid containmentstructure, such as a laminating adhesive layer. Useful cover layers forsensors having an active fluidic delivery system have inlet holes andoutlet holes for conducting the fluid sample through the sensor,optionally also including inlet and outlet flow connectors.

Fluid containment structures and photonic crystal structures of thepresent disclosure may comprise a wide range of materials includingpolymers such as mechanically flexible polymers. In embodiments usefulfor mass manufacture of disposable plastic sensors, the gratingstructures of the photonic crystal structure and the fluid containmentstructure are in the form of a monolithic, flexible polymer structurethat is fabricated via molding or imprinting techniques. Use of apolymer material for integrated fluid containment and photonic crystalstructures having a refractive index less than or equal to about n=1.6is beneficial for some applications. In some embodiments, sensors of thepresent disclosure further comprise a supporting substrate, such as apolymer, glass, ceramic or composite substrate, provided to the sensorso as to support the fluid containment structure and the photoniccrystal structure. Incorporation of a rigid substrate enhances thestructural rigidity and flatness of the sensor to facilitate handlingand optical readout of some sensors of the present disclosure.

Use of an at least partially optically transparent supporting substrateand/or rigid substrate is beneficial for some embodiments as this allowsfor optical read out by illuminating the bottom of the photonic crystalstructure. In some embodiments, integrated fluid containment structuresof the present disclosure are operationally connected to a mechanicalsupport structure, such as a bottomless microplate frame, for exampleprovided in a well microplate configuration, such as a standard 384 or1536 microplate configuration to further increase the volume availablefor a sample.

In some embodiments, sensors of the present disclosure comprise aphotonic crystal structure that is functionalized by incorporation oftarget material conjugated to an active surface of the photonic crystalsuch that the target material is exposed to the cavity of the fluidcontainment structure. In these embodiments, a target material may beprovided having selective binding characteristics so as to provideselective detection and analysis of specific analytes present in a fluidsample. In these embodiments, binding of analyte to a target materialconjugated to the active surface of the photonic crystal causes a changein refractive index in a probe region, thereby affecting the coupling ofelectromagnetic radiation into the photonic crystal and resulting in achange in photonic band gap. Useful target materials for biosensingapplications include, but are not limited to, one or more: proteins,peptide, DNA molecules, RNA molecules, oligonucleotides, lipids,carbohydrates, polysaccharides; glycoproteins, lipoproteins, sugars,cells, bacteria, virus, candidate molecules and all derivatives,variants and complexes of these. As will be apparent to those skilled inthe art, the target material may be conjugated to photonic crystalstructures using a variety of techniques and linking systems know in theart of sensing and biosensing.

The present disclosure encompasses sensor arrays and sensing systemswherein a plurality of sensors is provided, wherein each sensor hasindividual integrated fluid containment and photonic crystal structures.In some embodiments, a plurality of fluid containment structures andphotonic crystal structures are provided that comprise a singlemonolithic structure. In an embodiment, a plurality of sensors isprovided that comprise sensing and active fluidic delivery components ina multichannel sensing systems. Alternatively, the present disclosureincludes embodiments wherein a plurality of sensors is provided thatmake up sensing and fluid containment components in a multiwell arraysystem. An advantage of the present sensors and related fabricationmethods is that they may be provided in proximity to each other in adense area format useful for lab-on-a-chip devices, multichannel sensingsystems and microarray applications.

In another aspect, the present disclosure provides methods of makingphotonic crystal sensors having an integrated fluid containmentstructure. In one embodiment, a method of making a photonic crystalsensor having an integrated fluid containment structure comprises thesteps of: (1) providing a master template having an external surfacewith a pattern comprising (a) a photonic crystal periodic surfacegrating structure and (b) structure for forming a fluid containmentstructure, the periodic surface grating structure located within thestructure forming the fluid containment structure; (ii) transferring thepattern of the master template to a material such that the materialforms a fluid containment structure having a cavity with the photoniccrystal periodic surface grating structure positioned within the cavity;and (iii) depositing a thin dielectric film on the photonic crystalperiodic surface grating structure to thereby forming a photonic crystalsensor. In an embodiment, the material is a polymer, such as amechanically flexible, UV curable polymer. The fluid containment andphotonic crystal structure are a monolithic structure (part of the samecontinuous polymer material) and are fabricated simultaneously.Deposition of thin dielectric films may be carried out by any meansknown in the art including chemical and physical thin film depositiontechniques, such as magnetron sputtering, ion beam sputtering, plasmaenhanced chemical thin film deposition, electron beam evaporation andthermal evaporation.

The manufacturing process may include replica molding process in whichthe transferring the pattern on the master grating basically forms anegative of the surface on the master template on the material. Aperiodic grating structure pattern having selected physical dimensionson the grating master is transferred to the material. Alternatively, themanufacturing method encompasses methods in which the pattern transferis carried out using imprint lithography methods. Molding and imprintingfabrication methods of the present disclosure enable low cost, highthroughput fabrication of photonic crystal arrays and sensing systemsover very large areas (e.g., as large as 1 square foot at one time, upona continuous roll of flexible substrate that may be thousands of metersin length). Methods of the present disclosure using replica molding forpattern transfer are beneficial because these methods do not requiresignificant application of force to the external surface of the mastertemplate during pattern transfer, thereby avoiding damage to ordistortion of grating structures in the master template relief pattern.This attribute of the present disclosure allows for repeat processingusing a single master template and enhances pattern transfer fidelity.Use of polymer replica molding techniques are particularly beneficial asthey can be carried out at room temperature and may be performed uponflexible and optically transparent substrates in a continuousroll-to-roll fashion.

Patterning of the master template to generate the periodic surfacegrating structure of the photonic crystal and the structures for formingfluid containment structures may be carried out by any means known inthe art including deep UV optical lithography, E-beam writing,conventional optical lithography, optical write lithography, andmicromachining. In some methods, the master template is generated byprocessing of a semiconductor wafer via a two step top down processingprocedure, wherein nanosized grating structure features corresponding toa photonic crystal structure and a microsized a fluidic containmentstructures are defined in separate processing steps. In a firstprocessing step, an external surface of the wafer is patterned withphotoresist and etched so as to generate an external patterned layerhaving nanosized features provided in a spatially periodicconfiguration. This first processing step may be carried out, forexample, using deep-UV lithography and reactive ion etching. In a secondprocessing step, the external patterned layer of the semiconductor waferis subsequently processed so as to define the structures that form thefluid containment structure. This secondary processing step may becarried out using conventional optical lithography and deep reactive ionetching.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic diagrams illustrating a cross section view(FIG. 1A) and a top plan view (1B) of a biosensor of the presentdisclosure having an integrated fluid containment structure and aphotonic crystal sensor.

FIGS. 1C-1F are schematic diagrams illustrating several configurationsfor integrating a photonic crystal biosensor structure with arrays offluid containment cuvettes arranged in a standard 96-well microplateformat.

FIG. 2A is a process flow diagram illustrating an exemplary method forfabricating a sensor having an integrated fluid containment structureand a photonic crystal sensor. FIG. 2B is a schematic diagramillustrating a prior art method of making a photonic crystal sensorusing replica molding. FIG. 2C is a schematic diagram illustrating thepresent method for making integrated fluid containment and photoniccrystal structures using replica molding.

FIG. 3A is an illustration of a fabrication process used to produce abiosensor having microfluidic flow channel and photonic crystalpositioned within the flow channel. FIG. 3B is a scanning electronmicrograph of microfluidic channels with embedded photonic crystalsensors.

FIG. 4 is a schematic diagram of a representative imaging readoutinstrument for use with the biosensors of this disclosure.

FIG. 5 is an illustration of peak wavelength value (“PWV”) data gatheredby the imaging instrument of FIG. 4 of a biosensor having three fluidchannels incorporating a photonic crystal sensor, the three fluidchannels filled with deionized (DI) water. FIG. 5A shows the spatial PWVimage of the three channels. PWV shifts are represented by the scale barranging from 870 nm to 880 nm. FIG. 5B are graphs of sample reflectionspectra from each of the three channels. FIG. 5C is a horizontalcross-section plot showing PWV data along the green horizontal crosssection line in FIG. 5A. FIG. 5D is a vertical cross-section plotshowing PWV data along the vertical orange cross section line in FIG. 5a.

FIG. 6A is an illustration of the PWV shift measured by flowing in 6.25%dimethyl sulfoxide (DMSO) solution through channels 1 and 3, and flowingin DI water through channel 2 to serve as reference. PWV shifts arerepresented by the scale bar from −0.20 to 2.70 nm, where the red regionrepresent areas of greater positive shift. FIG. 6B is a plot of PWVshift measured with DMSO solution with concentration ranging from 0% to25%, in which the data points were linearly fitted with least squareapproximation with R² value equal to 0.996.

FIG. 7 a is a shifted PWV image (subtraction of the PWV image of proteinA coated channels from PWV image with channels 2 and 3 bound with IgGmolecules). The amount of shifts are represented by the scale bar from−0.60 to 1.65 nm, where red regions represent areas of greatest positiveshift. FIG. 7 b is a cross-sectional PWV shift plot along lines 1, 2 and3 for each of the three channels for PBS buffer, chicken IgG, and pigIgG respectively.

FIG. 8 is an illustration of a biosensor having integrated fluidcontainment structure (fluid flow channels) and photonic crystal sensorpositioned within the fluid flow channels.

FIG. 9 is a top plan view of a microplate configuration for a biosensorhaving a fluid channel system for delivery of fluid samples tomicrowells having photonic crystal structures at the bottom thereof. Thefluid channels also have photonic crystal sensors in the bottom surfaceof the fluid channels.

FIG. 10 is a perspective view of a plate configuration of a biosensor inthe form of a microfluidic cartridge.

FIG. 11 is a schematic diagram illustrating the exemplary processingsteps illustrating how the present sensor system can be used in onescenario using the biosensor of FIG. 10.

DETAILED DESCRIPTION

Biosensors are described herein which include one or more integratedfluid containment structures and a photonic crystal sensor, in amonolithic structure, e.g., a monolithic polymer structure. Fabricationmethods for making biosensors and integral fluid containment structureswill also be described below.

FIG. 1A is a schematic diagram illustrating a cross sectional side viewof a biosensor 100 of the present disclosure having an integrated fluidcontainment structure 110 and photonic crystal sensor 120. FIG. 1A isnot drawn to scale. The sensor shown in FIG. 1A can be considered as oneunit cell and replicated in the X direction. The sensor extends into thepage in the Y direction for some distance and the unit cell may repeatin the Y direction. The sensor is shown formed as a monolithic, layeredstructure. The base layer 200 is a substrate material, preferably andoptically clear material such as polyethylene terepthalate (PET), andlayer 105 is a clear polymer material such as UV curable epoxy.

The fluid containment structure 110 includes a cavity 130, which maytake the form of a channel in a microfluidic system or a staticreservoir such as a microwell in a micro array system. Cavity 130 has abottom internal surface 140 and internal sides 150. Photonic crystalsensor 120 is provided on the bottom internal surface 140 of the cavity130. As shown in FIG. 1A, the photonic crystal sensor 120 comprises agrating structure consisting of alternating high and low regions 160 and170, respectively, provided in a one dimensional spatially periodicconfiguration, collectively forming a 1-D a linear grating structure.The grating structure 160/170 can be one dimensional (periodic in onedimension) or periodic in two dimensions, such as in the form of anarray of posts or holes extending in the X and Y directions.Alternatively, the grating structure can take the form of 2-D, two-levelgrating.

A thin film 180 of a relatively high index of refraction material, suchas a dielectric or semiconductor film, is provided on high and lowstructures 160 and 170. Optionally, thin films 180 are also provide onside surfaces of grating structures 160 and on side internal surfaces150 of cavity 130 of the fluid containment structure 110. In a typicalembodiment, the thin film 180 is a TiO₂ or Ti₂O₃ layer which isdeposited onto the grating structure.

The grating structure 160/170 is formed in the material 105 in amanufacturing process to be described later on, such as for example areplica molding process using a grating master.

As shown in FIG. 1A, the grating structure 160/170 of the photoniccrystal sensor 120 and the fluid containment structure 110 areconstructed as a single monolithic structure, such as a monolithicpolymer structure, in contrast to prior art in which a photonic crystalwas manufactured separately and then fastened to another device such asa microwell plate. This monolithic structural configuration providessensors having precisely aligned fluid containment structures andphotonic crystal structures.

The substrate 200 can take the form of a polymer, ceramic or glasssubstrate, positioned to support the integrated fluid containmentstructure 110 and the photonic crystal structure 120.

Optionally, the sensor 100 further includes cover layer 210 positionedso as to enclose cavity 130 of the fluid containment structure 110. Thecover layer 210 may be fastened to the fluid containment structure 110by an adhesive layer 220, such as a laminating adhesive layer, andoptionally may have an inlet 240 and inlet flow connector 260 and anoutlet 230 and outlet flow connector 235 providing a means of flowing afluid sample through the sensor 100. The arrows provided in FIG. 1Aillustrate the flow of fluid sample through the sensor 100.

FIG. 1B shows a top plan view of the sensor 100 of FIG. 1A (not drawn toscale), with the cover layer 210 and adhesive layer 220 omitted. Thefluid containment structure 110 is in the form of a microfluidic channelhaving a photonic crystal sensor on its internal bottom surface. For thesake of illustration, the thin films 180 on top surfaces of high and lowgrating structures 160 and 170 are omitted. Also shown in FIG. 1B areinlet flow connector 260 and outlet flow connector 235. The fluidcontainment structure 110 could also be considered to take the form of avessel or well in which the sample to be tested is introduced into thewell via the inlet connector 260 and removed via the outlet connector235. In a variation, the fluid containment channel of FIG. 1B can extendin the Y direction and then change direction, e.g., be constructed toallow a fluid sample to flow along the fluid channel in a serpentinepath (see FIG. 8 for example).

FIGS. 1C-1F are schematic diagrams illustrating several configurationsfor integrating a photonic crystal biosensor structure with an array offluid containment cuvettes arranged in a standard 96-well microplateformat, but manufactured as a single monolithic integrated structure. Aswith fluid channels intended for dynamic flow of fluid past and over thephotonic crystal sensor surface, the cuvette fluid containmentreservoirs are fabricated with a similar process that also produces atleast one photonic crystal sensor on an internal surface of the cuvettefluid containment reservoir.

FIG. 1C shows a top view of a sensor array 300 comprising a plurality ofbiosensor cuvette sensors 100 each having integrated fluid containmentand photonic crystal sensors. Each cuvette sensor 100 may have thegeneral construction of FIGS. 1A and 1B. FIG. 1D shows a cross sectionalview of the sensor array 300 showing the cured replica molded biosensorcuvette sensors 100 supported by an optically transparent plasticsubstrate 200. FIGS. 1C and 1D are not drawn to scale. As shown in FIG.1D, each of biosensor cuvette sensors 100 comprises a fluid containmentstructure 330 (walls for holding a sample) and having a photonic crystalsensor 120 provided on its bottom internal surface. In some embodimentsthe fluid containment structures 330 have microsized physical dimensions(e.g., length, width and heights on the order of 10 s or hundreds ofmicrons), and the grating structures of photonic crystal sensors 340having nanosized heights and width (on the order of 10 s or 100 s ofnanometers), and micron-sized lengths in a one dimensional lineargrating configuration.

FIG. 1E is a cross sectional side view of an embodiment wherein a clear,rigid substrate 350 is further provided to support plastic substrate 200of the sensor array 300 of FIGS. 1C and 1D. The substrate 350 addsstructural integrity and facilitates handling of the sensor array.Embodiments incorporating a rigid substrate 350 also maintain flatnessof the photonic crystal structure, which is beneficial for ensuringreliable optical readout of the sensors.

FIG. 1F is a cross sectional side view of an embodiment in which thefluid containment volume provided by the containment structures 330 isincreased by incorporation of a bottomless microplate frame 360 to theupper surfaces of the containment structures 330. The deviceconfigurations illustrated in FIGS. 1C-1F may be extended to sensorarrays having any number of reservoirs, including standard 384 and1536-well microplate configurations.

FIG. 2A provides a process flow diagram illustrating an exemplary methodfor fabricating a biosensor in which the fluid containment and photoniccrystal sensors structures are fabricated simultaneously, i.e., in thesame manufacturing process. As shown in step 400 of this Figure, thefirst step is manufacture of a master template for use in a replicamolding process. This step includes sub-steps of: (i) providing asilicon wafer; (ii) processing the external surface of the silicon wafervia deep-UV lithography to generate a periodic surface grating structure(e.g., a 1 or 2-D periodic grating). The periodic surface grating isused to form a photonic crystal having the surface grating structure160/170 shown in FIG. 1 in the material 105. The step also includessub-step (iii) of further processing the external surface of the wafervia conventional optical lithography to generate at least onemicron-sized feature which forms the fluid containment structure, thefluid containment structure incorporating (surrounding) the photoniccrystal grating structure.

In step 402, the patterned surface of master template form in step 400is contacted with curable material. Step 402 includes sub-steps of: (i)providing the master template having the patterned external surface;(ii) contacting the patterned external surface of the master templatewith UV curable epoxy and (iii) allowing the UV curable epoxy to conformto the shape of features provided on the patterned external surface ofthe master template. The epoxy may be sandwiched between the patternedexternal surface of master template and a polymer (e.g., PET) substrate(substrate 200 in FIG. 1).

In Step 404 of FIG. 2A, the curable material is cured and released fromthe master template. This step includes sub-steps of (i) exposing theliquid UV curable epoxy in contact with patterned external surface oftemplate to ultraviolet electromagnetic radiation to cure the UV curableepoxy, thereby generating a patterned polymer layer in contact with themaster template; and (ii) peeling away the patterned UV material fromthe master template, thereby resulting in an integrated monolithicstructure having a fluid containment structure and photonic crystalsensor.

In step 406 of FIG. 2A, thin films of high index of refraction material(180 and 190 of FIG. 1) are deposited on to the photonic crystalstructure. For example, a layer of TiO₂ with a thickness of between50-500 nm is deposited on the high and low surfaces of the periodicgrating structure of the photonic crystal. The thin films may also beposited on the top surface of the fluid containment structure and theside walls as shown in FIG. 1A. The depositing of the thin dielectricfilm may, for example, be done by use of electron beam evaporationdeposition or other suitable process.

In step 408 of FIG. 2A, the fluid containment structure is optionallysealed with of a cover layer, and optionally providing inlet and outletports in the cover layer. The cover layer can be adhered to thestructure by means of an adhesive layer between the fluid containmentstructure and the cover layer. Inlet and outlet holes are then formed inthe cover layer and the adhesive layer. Additional features such astubes or ports or other similar structures can be added to facilitateattachment of vacuum, pump or injection devices to supply the sample tothe sensor.

FIG. 2B provides a schematic diagram illustrating a prior art method ofmaking a photonic crystal sensor using replica molding. As shown in thisFigure, a silicon master template 500 is provided having siliconsubstrate 502 and an external surface 504 with a plurality of gratingstructures 506 corresponding to the grating structures of the photoniccrystal sensors to be fabricated. In an embossing step, a layer of UVcurable material 105 is applied to the external surface 504 of thetemplate and allowed to conform to the shape of the grating structuresin the master template 500. The layer of UV curable material 105 incontact with the external surface of the master template is alsocontacted with a PET backing layer 200. UV light is directed onto thematerial 105. The UV curable material 105 is cured and then removed fromthe silicon master 500. The resulting product is an array of photoniccrystal structures.

FIG. 2C provides a schematic diagram illustrating the present method formaking integrated fluid containment and photonic crystal structuresusing replica molding. As shown in FIG. 2C, the silicon master templateis additionally patterned (etched) with structures 516 on its externalsurface corresponding to fluid containment structures, which in thisexample comprise a pair of fluidic channels 520. The structures 516 arelow regions which become raised structures in a UV curable materialwhereas the regions 518 are high regions which become channels forallowing fluid to flow into and over the photonic crystal gratingstructure 506. As shown in the bottom panel of FIG. 2C, incorporation ofthe additional structures 516 results in simultaneous formation of thefluidic channels (fluid containment structures) and the photonic crystalstructure upon the completion of the replica molding process.Furthermore, incorporation of the additional structures 516 provides forautomatic and high precision alignment of the fluid containment channels520 and the photonic crystal structures 506.

EXAMPLE 1 Single-Step Fabrication and Characterization of PhotonicCrystal Biosensors with Polymer Microfluidic Channels

Introduction

A method for simultaneously integrating label-free photonic crystalbiosensor technology into microfluidic channels by a single step replicamolding process is presented in this Example as one possibleimplementation of the disclosure.

By fabricating both the sub-micron features of the photonic crystalsensor structure and the >10 μm features of a flow channel network inone step at room temperature on a plastic substrate, the sensors areautomatically self-aligned with the flow channels, and patterns ofarbitrary shape may be produced. By measuring changes in the resonantpeak reflected wavelength from the photonic crystal structure induced bychanges in dielectric permittivity within an evanescent field regionnear its surface, detection of bulk refractive index changes in thefluid channel, or adsorption of biological material to the sensorsurface is demonstrated. An imaging detection instrument is alsodescribed which characterizes the spatial distribution of the photoniccrystal resonant wavelength, gathering thousands of independent sensorreadings within a single fluid channel.

Recently, microfluidic lab-on-a-chip (LOC) devices andmicro-total-analysis systems (μTAS) have been investigated in an effortto advance and simplify complex biochemical detection protocols forgenomics, proteomics, pharmaceutical high-throughput compound screening,and clinical diagnostic/biomedical applications on a small chip. Theneed for an automated μTAS to measure large numbers of biochemicalinteractions is currently being driven by industries and biologicalresearch worldwide. To operate a microfluidic system and carry out largenumbers of complex biochemical protocols, incorporation of sensors forfeedback control and detection of biochemical interactions for processmonitoring and verification is practically essential. This disclosureprovides sensors which meet these requirements.

For the majority of assays currently performed, fluorescent orcalorimetric chemical labels are commonly attached to the moleculesunder study so they may be readily visualized. However, using labelsinduces experimental uncertainties due to the effect of the label onmolecular conformation, blocking of active binding epitopes, sterichindrance, inaccessibility of the labeling site, or the inability tofind an appropriate label that functions equivalently for all moleculesin an experiment. Therefore, the ability to perform highly sensitivebiochemical detection without the use of fluorescent labels wouldfurther simplify assay protocols, and would provide quantitative kineticdata, while removing experimental artifacts from fluorescent quenching,shelf life and background fluorescence phenomena. While label-freebiosensors have been incorporated within separately attached flowchannels in the past, most systems are linked to a small number ofindependent sensor regions. What is needed is a sensor that enableshighly parallel detection of biochemical interactions with a high areadensity of independent sensors that can function without crosstalk.Ideally, such a system could be easily integrated with a fluid flownetwork without the need to align the sensors with the flow channels.Ultimately, sensors distributed throughout a chip will be capable ofmonitoring hundreds of biochemical interactions, and providing real-timefeedback to an integrated flow control system.

Previously, label-free optical biosensors based upon a sub-wavelengthphotonic crystal structure have been demonstrated. Because the photoniccrystal structure does not allow lateral propagation of resonantlycoupled light, a single photonic crystal surface is capable ofsupporting a large number of independent biosensor measurements withoutoptical crosstalk between adjacent sensor regions. Using an image-basedsensor readout method, we have demonstrated biosensor image pixelresolution as low as 9×9 μm², and have applied the imaging method todetect microarray spots, individual cells, and self-referenced assayswithin 96-well micro-plates. The photonic crystal surface has beenproduced over large surface areas from continuous sheets of plasticfilm, and has been incorporated into single-use disposable 96, 384, and1536-well micro-plates (all of which can be imaged for biochemicalbinding density at 9×9 μm² pixel resolution over their entire surfacearea).

In this example, we present for the first time a novel technique forintegrating label-free photonic crystal biosensor technology intomicrofluidic networks by replica molding photonic crystal sensors andfluid channels simultaneously. This approach enables detectionmodalities such as label-free biochemical detection, sample bulkrefractive index detection, and fluid presence within microchannels. Byfabricating multiple parallel channels in close proximity, highthroughput biochemical assays are enabled. Accurate correction ofcommon-mode error sources such as temperature and bulk solutionrefractive index variability is enabled by using sensors embedded in oneof the parallel channels as a reference.

The single step integration of photonic crystal biosensor structuresinto microfluidic channels presented here is also performed uponflexible plastic substrates using a replica molding approach to enable asimple low-cost manufacturing process to produce sensors and flowchannels of arbitrary shape that are automatically aligned to eachother. Disposable plastic chips would be less expensive than reusableglass devices and would eliminate time-consuming regeneration steps. Inaddition, the polymer used for the molded structure has superior solventresistance and gas permeability properties as compared topolydimethylsiloxane (PDMS), where incompatibility with most organicsolvents has limited its use to aqueous-based applications. Finally,through the use of an image-based detection approach, this system iscapable of observing the spatial profile of biochemical binding withinthe fluid channel, both across the channel width, and along the channellength.

Materials and Methods

1. Microfluidic Sensor Fabrication

The fabrication process requires a method that can accurately producesub-micron features for the photonic crystal structure, while at thesame time 30 producing the >10 μm features of the microfluidic channel.A replica-molding process using a rigid “master” structure and aUV-curable liquid polymer material was selected for this purpose becausethe molding may be performed at room temperature without the requirementto exert a large force between the mold and the molded material.

An outline of the fabrication procedure is shown in FIG. 3 a. First, asilicon master wafer 500 with 550 nm period 1-D linear gratingstructures 506 was fabricated. The grating structures 506 were patternedwith photoresist using deep-UV lithography, in which 6.7 mm diametercircular dies were stepped and repeated every 9 mm. After the exposedphotoresist was developed, the patterned grating structure wastransferred to the silicon wafer by reactive ion etch to a depth ofapproximately 170 nm. After etching, the photoresist was removed. Next,the fluid channel structures 516 were patterned onto the same siliconmaster wafer with grating structures from the previous step usingphotoresist again, but with conventional lithography. Because highresolution is not required for defining the channels (channel widths of30-250 μm were investigated), and to maximize flexibility forinvestigating different channel shapes, the photomask for the channelpatterns was produced upon a transparent plastic sheet with 5080 dpihigh resolution printing. After developing the exposed photoresist,channel structures 516 were transferred onto the silicon wafer usingdeep reactive ion etch with depth of approximately 20 μm, followed byremoval of photoresist. As a result, a negative pattern template ofmicrofluidic channels incorporated with sub-micron scale linear gratingstructures was formed. Subsequently, the completed silicon template wastreated with repel silane (GE Healthcare) to promote clean release ofreplica from the template without contaminating the template structureswith polymer residues.

Utilizing the silicon master wafer as a mold, the surface structure506/516 of the master wafer 500 was replicated onto a 250 μm thickflexible polyethylene terephthalate (PET) substrate 200 by distributinga layer of liquid UV curable polymer 105 between the silicon masterwafer 500 and the PET substrate 200. The liquid polymer conforms to theshape of the features on the master wafer, and is subsequently cured toa solid state by exposure to UV light 600. After the polymer was cured,the surface structure was peeled away from the silicon wafer, leavingbehind a replica of the silicon master wafer surface adhered to the PETsheet (FIG. 3 (iv)). The sensor was completed by depositingapproximately 150 nm of titanium dioxide (TiO₂) shown as layer 180 inFIG. 3 (v) using electron beam evaporation on the replica surface. TheScanning Electron Micrograph (SEM) images in FIG. 3 b show the curedreplica surface coated with TiO₂, in which the replicated flow channel520 contains the photonic crystal biosensor 120 on its bottom surface.

The upper surface of the microfluidic channel 520 was completed bysealing with a separate PET sheet 210 with inlet and outlet holes, usinga layer of 2-sided pressure-sensitive adhesive film 220 (3M) in between(FIG. 3, part (vi)). The sealed plastic microfluidic sensor chip wasthen attached with the same transparent film adhesive to the surface ofa 1×3 square inch glass microscope slide to provide structural rigidity.The microfluidic sensor chip was completed by attaching polypropylene(PP) flow connectors on the inlet holes of the PET cover layer usingadhesive, followed by reinforcement sealing with clear epoxy. Flowingfluids into the microfluidic channels 520 was accomplished bypre-filling the PP flow connectors with solutions or analytes andmanually pumping it using a syringe with tubing connected to PP flowconnector. Manual syringe pumping method was sufficient becauseexperiments performed in this work involved filling the channels withsolutions, incubating/stabilizing at room temperature, washing/rinsingwith buffer, and therefore were independent of fluid flow rate.

2. Imaging Instruments

As will be recognized by those skilled in the art, a great variety ofoptical illumination, analysis and detection systems may be used inconjunction with the present sensors, for example as described in thepreviously-cited patent literature. Such instruments will typicallyinclude suitable illumination apparatus, and optical and detectioncomponents so as to enable optical read out, including read out inoptical imaging and point detection modes. The instruments include alight source positioned in optical communication with the sensor suchthat the photonic crystal structure is illuminated with electromagneticradiation having a selected wavelength distribution, for exampleelectromagnetic radiation having a wavelength distribution in thevisible, ultraviolet or infrared regions of the electromagneticspectrum. A photodetector is positioned in optical communication withthe photonic crystal structure such that it is capable of analyzing anddetecting electromagnetic radiation reflected, scattered or transmittedby the photonic crystal structure. Useful optical sources include broadband sources, including quartz lamps, xenon lamps, halogen lamps and/ordeuterium lamps. Useful photodetectors comprise optical analyzersincluding dispersive elements, such as spectrometers, gratings andprisms, and optical detectors such as photomultiplier tubes,photodiodes, diode arrays and CCD imaging systems.

In one possible embodiment, the optical source is a broad band source incombination with a polarization filter that provides electromagneticradiation at normal incidence to the sensor having a polarizationdirection perpendicular to grating lines of the photonic crystalstructure. A beam splitter and imaging lens is provided to collectelectromagnetic radiation reflected from the sensor and direct it to theaperture of a spectrometer. Detection is carried out using a twodimensional detector, such as a CCD camera. In this optical read outconfiguration, electromagnetic radiation from a line on the photoniccrystal structure is wavelength analyzed and detected, optionally as afunction of time. Spectral analysis provided by this detectionconfiguration provides a spatially resolved spectrum for each pointwithin the line, thereby allowing determination of the wavelengthdistribution, and optionally peak wavelength for each point on the line.The detection system may further include a motorized stage capable oftranslating the sensor such that two dimensional images of the photoniccrystal structure are obtained. Alternatively, the detection instrumentmay include optical instrumentation capable of scanning the illuminatingbeam of electromagnetic radiation over selected regions of the sensorsuch that two dimensional images of the photonic crystal structure areobtained.

A schematic diagram of a biosensor peak wavelength value (PWV) imaginginstrument used in Example 1 is shown in FIG. 4. The instrument includesa light source 610, mirror 612, beam splitter 614, a polarizing filter616 and an imaging spectrometer 620. White light from the light source510 illuminates the sensor 100 at normal incidence, with a polarizationfilter 616 to only allow passage of light with polarization directionperpendicular to the sensor grating lines. The reflected light isdirected through the beam splitter 614 to an imaging lens of unitymagnification (not shown) and to a narrow entrance slit aperture 622 ofan imaging spectrometer 620. The width of the slit 622 may be set at adesired value, e.g. within a range from 6 to 200 μm. Using this method,reflected light is collected from a line on the sensor 100 surface,where the width of the imaged line is determined by the width of theentrance slit 622 of the imaging spectrometer. The imaging spectrometer620 contains a two-dimensional CCD camera (Acton Research) with 2048×512pixels. The line of reflected light, containing the biosensor resonancesignal, is diffracted by a diffraction grating in the spectrometer 620to produce a spatially-resolved spectrum from each point within theline. When the CCD camera is operated in 2048×512 pixel mode, theline-image through the slit is imaged onto 512 pixels. A spectrum, witha resolution of 2048 wavelength data points, is acquired for each of the512 pixels. Upon peak-finding analysis of all 512 spectra, the PWVs of512 pixels are determined. Thus, a line 628 of 512 pixels is generatedfor the PWV image 630 of the sensor.

To generate a two-dimensional PWV image of the sensor, a motorized stage(not shown) translates the sensor 100 which is placed on a preciseholding fixture, in the direction that is perpendicular to the imageline. See arrow 632 in FIG. 4. The spatial separation of the image linesis determined by the step-size of the stage between each image-lineacquisition (In addition, the CCD can be read out with variousresolutions by binning pixels). By this technique, a series of lines areassembled into an image through software and same spot in the sensor canbe scanned repeatedly after the sensor has been translated. In thecurrent system, the length of the image line is 9.1 mm, as determined bythe size of the CCD chip, across the biosensor surface. A large area canbe scanned in a tiled fashion, where the width of a tile is 9.1 mm, bytranslating the sensor in steps of 9.1 mm along the image-linedirection.

Typically, a biosensor experiment involves measuring shifts in PWV, sothe sensor surface is scanned twice, once before and once afterbiomolecular binding, and the images are aligned and subtracted todetermine the difference in PWV as detected by the sensor. This scanningmethod does not require the PWV of the imaged surface to be completelyuniform, either across the surface or within a set of probe locations,or tuning of the sensor angle to a resonance condition as with SurfacePlasmon Resonance (SPR) imaging.

Results and Discussion

1. Bulk Refractive Index Sensitivity Experiment

The sensor structure integrated within the fluid channels measureschanges in dielectric permittivity upon its surface. Therefore, flowingliquid solutions with variable refractive index through the fluidchannels induces a PWV shift. Because refractive index correspondslinearly with dimethyl sulfoxide (DMSO) concentration in deionized (DI)water, the dependence of PWV on bulk refractive index was determined byflowing in different concentrations of DMSO solution in DI water to thefluid channels.

In this experiment, a sensor 100 having three fluid channels, eachhaving its own photonic crystal sensor in the bottom thereof, was used.The three channels are designated p1, p2 and p3 in the followingdiscussion and in FIGS. 5 and 6.

Initially, all three channels were filled with DI water and a baselinePWV imaging scan at 22.3 μm resolution was made using the instrument.The resulting spatial PWV image is shown in FIG. 5 a, in which PWVs arerepresented by the scale bar 670 ranging from 870 nm to 880 mm, with redregion 680 representing areas of higher PWV. FIG. 5 b shows samplereflection spectra from one data pixel from each channels, with PWVs of877.79, 877.65, 876.87 nm for channels p1, p2 and p3, respectively.FIGS. 5 c and 5 d are cross section plots of the spatial PWV image. Theplot in FIG. 5 c represents PWVs along the green horizontal crosssection line 650 of FIG. 5 a, and likewise, FIG. 5 d represents PWVsalong the orange vertical cross section line 660 of FIG. 5 a. The crosssection PWV plots indicate that the PWVs vary slightly from differentchannels and even within the same channel (FIG. 5 c). This is acceptablesince quantity of interest in this case is the shift in PWV whendifferent solutions are introduced or some biochemical reaction occurson the sensor surface, rather than the PWV value itself.

After taking a PWV image scan with the channels filled with DI water,channels 1 and 3 were filled with DMS0 solution while channel 2 wasrefilled with DI water, to serve as a reference. FIG. 6 a shows aspatial PWV shift image measured by flowing in 6.2S % DMSO solutionthrough channels 1 and 3. Shifted PWV image is obtained by subtractingthe reference spatial PWV image with all channels filled with DI water(FIG. 5 a), from the spatial PWV image of the exact same device filledwith 6.2% DMSO solutions in channels 1 and 3.

Therefore, PWV variations caused by fabrication non-uniformity shown inFIGS. 5 a, c, and d does not result in significant sensitivitynon-uniformity as PWV image subtraction is performed. PWV shifts arerepresented by the scale bar 670 from −0.2 to 2.7 nm, where red regionsrepresent areas of greatest positive shift. The overall standarddeviation for shifted PWV of data was 0.263 nm.

Once the shifted PWV images are obtained, grids of sensor regions areselected (Square areas in FIG. 6 a), in which many independent pixelreadings within each grid can be averaged into a single measurement. Amasking function is applied so that only resonant peaks with reflectedintensity maxima above a user-selectable value are considered for theselection of spectra within the grid. Through the masking function,therefore, regions of the chip that do not contain a photonic crystalstructure (such as the regions between flow channels) that do notreflect a resonant peak, are automatically eliminated from furtherconsideration. Each grid can be designated as “active” or “reference”,and PWV shifts from reference regions can be associated with any desiredactive region for subtraction of common-mode artifacts. In thisexperiment, the PWV shift was calculated by subtracting the average PWVshift within the grid of channel 2 (reference), from the average PWVshift of the grids for channels 1 and 3 (active). Because of thedifferences in channel width (150, 200, and 250 μm for channels 1, 2,and 3 respectively), the number of independent data pixels satisfyingthe mask function within each grid for channels 1, 2, and 3 were 2560,4337, and 7509 respectively.

Scans were made after flowing in each of the different DMSOconcentrations ranging from 0.78% to 25% through channels 1 and 3. Bothchannels were rinsed with DI water and dried before flowing in differentconcentrations of DMSO solutions. FIG. 6 b plots the PWV shift as afunction of DMSO concentration, in which the data points were linearlyfitted with least square approximation with R² value equal to 0.996,showing the expected linear dependence between photonic crystalreflected resonant PWV and the solution bulk refractive index. Theapproximate bulk refractive index change corresponding to 6.25% changein DMSO concentration (Δ PWV of 1.841 nm) is 0.00682, based on the bulkrefractive index shift coefficient (σ=ΔPWV/Δn) value of 270, determinedfrom previous research.

2. Protein A—Immunoglobulin G (IgG) Experiment

An experiment was performed to demonstrate detection of biomolecularbinding on the surface of the photonic crystal sensor within the fluidchannels. Protein A (Pierce Biotechnology) was used as the immobilizedprotein ligand on the sensor surface, while chicken IgG and pig IgG(Sigma-Aldrich) were used as analytes. Pig IgG is known to have a strongbinding affinity for Protein A, while chicken IgG is known not to bindwith Protein A, and therefore acts as a negative control for ourexperiment.

Before immobilization of Protein A, a baseline PWV image of threechannels filled with PBS buffer (Sigma-Aldrich) was taken at a pixelresolution of 22.3 μm. The Protein A was attached by simple physicaladsorption by flowing a 0.5 mg/mL solution through all three channelsp1. p2 and p3, allowing the solution to incubate for 10 minutes,followed by washing away of unbound Protein A with PBS buffer. A secondPWV image was gathered after Protein A immobilization, with PBS bufferin the channels. Next, channel 1 was filled with PBS buffer to serve asa reference, while channels 2 and 3 were filled with 0.5 mg/mLconcentration solutions of chicken IgG and pig IgG respectively. TheIgGs were allowed to incubate with the immobilized Protein A for 10minutes, followed by a thorough PBS wash to remove unbound IgGs. Then, afinal PWV scan was made with all three of the channels filled with PBSbuffer

FIG. 7 a shows a PWV shift image for subtraction of the PWV image afterProtein A coating from the PWV image after IgG binding for all threechannels. PWV shifts are represented by the scale bar 670 from −0.60 to1.65 nm, where red regions 680 represent areas of greatest positiveshift. As shown in FIG. 7 a, three horizontal lines 700, 702 and 704within each channels (lines 1, 2 and 3 colored in orange, red and bluerespectively) are selected, in which independent PWV shift pixel dataalong the lines are sampled. The number of independent data pixelssampled within each line is 190.

FIG. 7 b is the cross sectional PWV shift plot along lines 1, 2 and 3(700, 702, 704) for each of the three channels for PBS buffer, chickenIgG, and pig IgG, respectively. In order to calculate the overall PWVshifts for the IgGs, square grids 706 of sensor regions, shown in FIG. 7a, are selected, in which many independent pixel PWV data within eachgrid 706 can be averaged. Again, because of the differences in channelwidth (150, 200, and 250 μm for channels 1, 2, and 3 respectively), thenumber of independent data pixels sampled within each grid for channels1, 2, and 3 were 2223, 5449, and 6208, respectively. For thisexperiment, the overall average PVVV shifts for IgGs were calculated bysubtracting average PWV shift within the grid of channel I which is thereference, from average PWV shift of grids for channels 2 and 3corresponding to chicken IgG and pig IgG, respectively. Using the abovemethod, the average PWV shift measured and calculated in the chicken IgGand pig IgG containing sensor channels were −0.051 and 0.815 nm,respectively, demonstrating selective attachment of the Pig IgG analyteto the immobilized Protein A.

FIG. 8 is an illustration of a biosensor suitable for use in performingthe experiments such as described above. The sensor 300 includes inletports 800 for introduction of a fluid sample. The sensor 300 featuresmicron-scale fluid channels 520 each containing a photonic crystalsensor as described above. Outlet ports 802 are provided which connectto vacuum apparatus (not shown) or to pumps or injection devicesallowing the sample to be drawn through the channels 520 and over thephotonic crystal sensor incorporated therein. The photonic crystalsensor and the fluid channels are part of an integrated, monolithicstructure which is fabricated using the molding process using the mastertemplate in the manner described previously.

Discussion

The fabrication and detection methods described in this work representthe building blocks that may be used to design and build moresophisticated lab-on-a-chip systems incorporating sensors for label-freebiochemical or cellular analysis. This work demonstrates that a narrowphotonic crystal region within a flow channel provides a strong resonantreflection signal, and that a large number of independent “pixels” maybe monitored at one time within a small chip. The imaging capability maybe utilized in several ways to improve the resolution and/or throughputof label-free measurements. As demonstrated with the serpentine flowchannel design, a single “line” of PWV measurements across the width ofmany flow channels may be used to monitor biochemical binding in a largenumber of flow channels at one time. Although only “end point”measurements were shown here, a single PWV line may be scanned rapidly(˜20 milliseconds per scan) to gather kinetic binding data for all theflow channels intersecting the line. Further, PWV measurements are notlimited to a single reading across the width of a flow channel, butrather the variability in binding density from the center to the edge ofthe channel is easily detected.

These types of measurements will enable optimization of flow conditionsand direct observation of edge effects that are not normally detected.Likewise, the serpentine flow channels allowed us to demonstratedetection of biochemical binding down the length of a single flowchannel, where again rapid scanning will allow direct observation ofimmobilized ligand density binding variability and detected analytevariability, and any nonuniformity resulting from mass transportlimitations. By taking many independent binding readings down the lengthof a channel, we expect to reduce the statistical (random) noise ofindividual PWV determinations to extremely low levels through averaging.In the case of our serpentine channel configuration, all the PWV shiftreadings, with >6000 readings within a single channel for ˜22×22 μm²pixels, are easily gathered together to calculate an average PWV shiftmeasurement for the entire channel.

The sensors of this disclosure allows reference channels to beincorporated in close physical proximity to active channels for highlyaccurate correction of temperature or buffer variability. Because activeand reference regions are small, many reference regions may be easilyincorporated onto a single chip.

The present disclosure also is compatible with more complex sensor/flowchannel configurations that incorporate valving and mixing capabilitiesinto the chip. This capability is useful not only for biochemicalassays, but also for detection of immobilization of larger biologicalobjects, including cells and bacteria for cytotoxicity assays,chemotaxis assays, and diagnostic tests, and cell/bacteriaidentification.

From the above disclosure, it will be appreciated that we havedemonstrated in this example a single-step process for integrating thefabrication of photonic crystal biosensors and microfluidic channels.The process enables the submicron structure of the photonic crystal tobe performed simultaneously with the >10 micron structures for the fluidchannels, and self-aligns the photonic crystal sensors with thechannels. The process can be performed using a room-temperature replicamolding process that is performed on flexible plastic substrates forlow-cost manufacturing. The fabricated sensors may be measured in ahigh-resolution imaging mode that can obtain information from manylocations within the chip surface simultaneously for monitoringbiochemical interactions in a high throughput manner and observation ofbinding interaction uniformity along the lengths and across the widthsof the channels. We demonstrated the ability of the integrated sensorsto detect changes in the bulk refractive index of fluid introduced intothe channels, and to selectively detect an antibody at highconcentration with an immobilized ligand. In the current work, flow wasused to introduce reagents to the sensors in the channels. Wedemonstrate capabilities for applications in pharmaceutical compoundscreening, protein-protein interaction characterization, and cell-basedassays using the presently described processes and structure uponincorporation of additional flow systems and elements.

EXAMPLE 2 Photonic Crystal Sensors with Fluid Containment StructuresHaving a Microplate Configuration

Introduction

The present disclosure also contemplates microplate sensor systemscomprising arrays of microwells, each having individually addressedphotonic crystal sensors. Microplate sensor systems of this aspect ofthe present disclosure may further comprise integral micron scale fluidcontainment structures (channels) for introducing fluid samplescontaining analytes into selected microwells.

FIG. 9 is a schematic, top plan view of a microwell configuration 900for a sensor system of this embodiment in the form of an array of 12×8microwells 920. The sensor includes a fluid handling system 910 in theform of fluid channels 940 for delivery of fluid samples from ports 930to the microwells 920. Each of the fluid channels 940 include photoniccrystal sensors 950. The bottom surface of the microwells 920 include aphotonic crystal sensor as shown the embodiment of FIG. 1A. The sensors950 allow label-free measurements (referred to herein occasionally as“BIND” measurements) of the sample, whereas the photonic crystal sensorsin the bottom of the microwells 920 allow measurements to be made afterthe sample flowing in the channels has been allowed to pass into thewells and interact with a second sample material added to themicrowells. This possible use of the sensor will be described in detailbelow.

The inset in FIG. 9 provides an expanded view of a portion of one columnof microwells and the corresponding fluid handling system 910. Featuresof this sensor include: (i) The eight microwells 920 in each column areattached to a common port 930 for both loading and waste; (ii) thephotonic crystal sensors 950 in the channels associated with each of theeight wells are in substantial alignment, and all fall on the samehorizontal line; (iii) The resistance (length) of the each channel 940among the eight wells is identical, both between the microwell 920 andthe photonic crystal sensor 950, and between the sensor 950 and thecommon port 930. This is achieved in the embodiment shown in FIG. 11 byuse of serpentine channels 960 to add extra length as required to makethe path length of the channels all the same.

Advantages of the system shown in FIG. 9 include (i) All the photoniccrystal sensors 950 are aligned and in one row, therefore only onedimensional scanning required to make measurements of bindinginteractions in the sensors 950; (ii) Each channel 940 has the samelength and fluid resistance, therefore the time for sample material tomigrate to the sensors and to the microwells is the same for all thewells in a column of wells; (iii) eight common ports 930, one port percolumn of wells, means that eight different receptors (analytes) addedto the wells can be tested in parallel.

FIG. 10 provides a schematic diagram of a perspective view of amicrowell configuration of this example in the form of a microfluidiccartridge 1000. As shown in this figure a PET supporting substrate layer200 is provided which supports a patterned polymer layer 105 comprisinga plurality of integrated fluid containment and photonic crystalstructures. The sample wells 920 are tapered to minimize the fluidvolume needed to fill them. An additional polycarbonate layer 360 isprovided to enhance the volume capacity of the microwells and addstructural integrity. (See the design of FIG. 1F).

Several device scenarios are contemplated for the embodiment of FIGS. 9and 10, including:

Scenario A: The twelve common wells 930 are all filled with the samereceptor protein, which enters all of the channels and binds to thephotonic crystal BIND sensors placed in the channels 940 leading to thewells 920. Then, 96 unique analytes are loaded directly into the samplewells 920, and 96 assays are performed simultaneously using photoniccrystal sensors contained in the sample wells 920. Alternatively, theuser may wish to do twelve unique assays, one per column of wells, witheight repetitions for each assay.

Scenario B: The twelve common wells 930 are each filled with a differentreceptor protein, thereby labeling each column of eight wells 920 with adifferent protein. Then, eight unique analytes are introduced across thetwelve rows, performing 96 simultaneous assays that test eight analytesversus twelve receptors.

Scenario C: Ninety six unique proteins are deposited in the sample wells920 and allowed to bind to the photonic crystal sensors positioned inthe bottom of the sample wells 920. One analyte is introduced into thetwelve common wells 930 and allowed to flow into the wells 920. Ninetysix unique assays are performed with the same analyte and differentreceptors.

The geometry can be changed so that the common wells (and the datareadout) run in the other direction, allowing Scenario B to be reversedto twelve analytes versus eight receptors.

FIG. 11 provides a schematic diagram illustrating the exemplaryprocessing steps illustrating how the present sensor system can be usedin Scenario A of this Example. For simplicity, only one common well 930and two sample wells 920 are shown in FIG. 11. Also note that the samplewells are not shown tapered, but use of tapered well is within the scopeof this embodiment. The common well 930 is connected to the sample wells920 via channels 940A and 940B, each having a photonic crystal sensor950 positioned in the bottom surface of the channel 940A and 940B.

Step 1 shows the sensor system with the device empty. Step 2 shows thesensor system with receptor protein 1100 loaded into the common well930. Step 3 shows receptor protein entering into channels 940A and 940Bthrough hydrostatic pressure and capillary action. The fluid moves farenough to cover the photonic crystal sensors 950 but not much further.The device is designed so that fluid flow stops at the appropriatedistance, in the channels 940 but past the photonic crystal sensors 950.Receptor molecules bind to the sensors 950 during this phase and a labelfree “BIND” PWV measurement is taken from the sensors 950 (e.g., usingthe instrument of FIG. 4). Step 4 shows the systems as two differentanalytes 1102 and 1104 are loaded into the sample wells 920. Step 5shows the system as analytes 1102 and 1104 are pumped into channels 940Aand 940B by hydrostatic pressure or vacuum until the height of thecolumns is equal in the sample wells 920 and the common well 930. Thisis designed so that sample flow proceeds past the sensors 950. Thebinding reactions between the analytes 1102 and 1104 and the receptormolecules bound to the photonic crystal sensor 950 starts and can bemeasured via PWV measurements in the manner described previously.

From the above discussion, it will be appreciated that we have describeda photonic crystal biosensor (100, 300, 1000) with a fluid containmentstructure (e.g., channels 940, or wells 130 or 920) having a cavity(well or channel), integrated with a photonic crystal sensor (120/950)comprising a periodic surface grating structure formed in the internalsurface or cavity of the fluid containment structure, as shown in thedrawings, wherein the fluid containment structure and periodic surfacegrating structure of the photonic crystal structure comprise a integral,monolithic structure.

As shown in FIG. 1A, the integral, monolithic structure may take theform of an an integral polymer structure comprising an optically clearsubstrate layer 200, a cured polymer layer 105, and a relatively highindex of refraction material 180 deposited on the cured polymer layer105.

As shown for example in FIGS. 9 and 11, the sensor may includes a cavityin the form a sample well 920, and wherein the integral monolithicstructure further comprises a port 930 for receiving the fluid sample, achannel 940 having a surface and providing a fluid path for connectingthe port 930 to the micron-scale fluid containment structure 920, and asecond photonic crystal structure 950 comprising a periodic surfacegrating structure formed in the surface of the channel.

In the above embodiments, the sensor may include a target material whichis bound to the periodic grating structure of the photonic crystalstructure, with the target material exposed within the fluid containmentstructure (well or channel) for binding of an analyte. Examples of thetarget material include proteins, peptides, DNA molecules, RNAmolecules, oligonucleotides, lipids, carbohydrates, polysaccharides;glycoproteins, lipoproteins, sugars, cells, bacteria, virus, andcandidate molecules.

GLOSSARY

“Polymer” refers to a molecule comprising a plurality of repeatingchemical groups, typically referred to as monomers. Polymers are oftencharacterized by high molecular masses. Polymers useable in the presentdisclosure may be organic polymers or inorganic polymers and may be inamorphous, semi-amorphous, crystalline or partially crystalline states.Polymers may comprise monomers having the same chemical composition ormay comprise a plurality of monomers having different chemicalcompositions, such as a copolymer. Cross linked polymers having linkedmonomer chains are particularly useful for some applications of thepresent disclosure. Polymers useable in the methods, devices and devicecomponents of the present disclosure include, but are not limited to,plastics, thermoplastics, elastomers, elastoplastics, thermostats, andacrylates. Exemplary polymers include, but are not limited to,polymethylmethacrylate, acetal polymers, biodegradable polymers,cellulosic polymers, fluoropolymers, nylons, polyacrylonitrile polymers,polyamide-imide polymers, polyimides, polyarylates, polybenzimidazole,polybutylene, polycarbonate, polyesters, polyetherimide, polyethylene,polyethylene copolymers and modified polyethylenes, polyketones,polymethylpentene, polyphenylene oxides and polyphenylene sulfides,polyphthalamide, polypropylene, polyurethanes, styrenic resins, sulphonebased resins, vinyl-based resins or any combinations of these.

The term “electromagnetic radiation” and “light” are used synonymouslyin the present description and refer to waves of electric and magneticfields. Electromagnetic radiation useful for the methods of the presentdisclosure includes, but is not limited to ultraviolet light, visiblelight, infrared light, microwaves, radio waves or any combination ofthese.

“Optical communication” refers to a configuration of two or moreelements wherein one or more beams of electromagnetic radiation arecapable of propagating from one element to the other element. Elementsin optical communication may be in direct optical communication orindirect optical communication.

“Direct optical communication” refers to a configuration of two or moreelements wherein one or more beams of electromagnetic radiationpropagate directly from a first device element to another without use ofoptical components for steering and/or combining the beams. “Indirectoptical communication” on the other hand refers to a configuration oftwo or more elements wherein one or more beams of electromagneticradiation propagate between two elements via one or more devicecomponents including, but not limited to, wave guides, fiber opticelements, reflectors, filters, prisms, lenses, gratings and anycombination of these device components.

“Thin film” refers to a coating or layer of atoms, molecules or ions ormixtures and/or clusters thereof. Thin films in the present disclosuremay comprise a single-layer having a substantially constant composition,a single-layer having a composition which varies as a function ofphysical thickness or a plurality of thin films layers. Thin film layersof the present disclosure include but are not limited dielectricmaterials, semiconductors, conducting materials, organic materials suchas polymers and any combinations of these materials. In a preferredembodiment, reference to thin dielectric films in the present disclosureincludes but is not limited to metal oxide, metalloid oxide and saltthin films. Thin film layers of the present disclosure may have anysize, shape, physical thickness or optical thickness suitable for aselected application.

The terms “frequency distribution of a photonic band gap” and“reflectance spectrum of a photonic band gap” are used synonymously inthe present description and refer to the frequencies of incidentelectromagnetic radiation that transmission through a photonic crystalis at least partially prevented. The present disclosure provides dynamicphotonic crystals having a tunable photonic band gap wherein thefrequency distribution of the photonic band gap may be selectivelyadjusted by exposure of the crystal to polarized excitationelectromagnetic radiation.

As used herein, “nanosized” refer to features having at least onephysical dimension (e.g. height, width, length, diameter etc.) rangingfrom a few nanometers to a micron, including in the range of tens ofnanometers to hundreds of nanometers. In an embodiment, a nanosizedfeature is structure, relief feature or relief feature having at leastone physical dimension that is on the order of hundreds of nanometer.For example, the width and/or height of a nanosized feature can be onthe order of 10's to 100's of nm and the length of a nanosized featureof can be on the order of microns to 1000's of microns.

As used herein, “micron-sized” refer to features having at least onephysical dimension (e.g. height, width, length, diameter etc.) rangingfrom a micron to a thousand microns, including in the range of tens ofmicrons to hundreds of microns. In an embodiment, a micron-sized featureis a structure having at least one physical dimension ranging from about1 micron to about 1000 microns. For example, the width and/or height ofa microsized feature can be on the order of 10's to 100's of microns andthe length of a microsized feature of can be on the order of millimetersto centimeters.

As used herein the term “fluid” refers to a material that is capable offlow and conforms, at least partially, to the outline of its container.Fluids in the present disclosure include liquids, gases, solutions,colloids (e.g., aerosols, emulsions, gels and foams) and anycombinations and mixtures of these. “Polymer layer” refers to a layerthat comprises one or more polymers. Polymer layers useful in thepresent disclosure may comprise a substantially pure polymer layer or alayer comprising a mixture of a plurality of different polymers. Polymerlayers useful in the present disclosure also include multiphasepolymeric layers and/or composite polymeric layers comprising acombination of one or more polymer and one or more additional material,such as a dopant or structural additive.

“Candidate molecules” include therapeutic candidate molecules which aremolecules that may have some effect on a biological process or series ofbiological processes when administered to a human, other animal or plantsubject. Therapeutic candidate molecules include, but are not limitedto, drugs, pharmaceuticals, potential drug candidates and metabolites ofdrugs, biological therapeutics, potential biological therapeuticcandidates and metabolites of biological therapeutics, organic,inorganic and/or hybrid organic inorganic molecules that interact withone or more biomolecules, molecules that inhibit, decrease or increasethe bioactivity of a biomolecule, inhibitors, ligands and derivatives,variants and complexes of these.

Where the terms “comprise”, “comprises”, “comprised”, or “comprising”are used herein, they are to be interpreted as specifying the presenceof the stated features, integers, steps, or components referred to, butnot to preclude the presence or addition of one or more other feature,integer, step, component, or group thereof. Separate embodiments of thedisclosure are also intended to be encompassed wherein the terms“comprising” or “comprise(s)” or “comprised” are optionally replacedwith the terms, analogous in grammar, e.g.; “consisting/consist(s)” or“consisting essentially of/consist(s) essentially of” to therebydescribe further embodiments that are not necessarily coextensive.

The disclosure has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the disclosure. It will be apparent toone of ordinary skill in the art that compositions, methods, devices,device elements, materials, procedures and techniques other than thosespecifically described herein can be applied to the practice of thedisclosure as broadly disclosed herein without resort to undueexperimentation. All art-known functional equivalents of compositions,methods, devices, device elements, materials, procedures and techniquesdescribed herein are intended to be encompassed by this disclosure. Thisdisclosure is not to be limited by the embodiments disclosed, includingany shown in the drawings or exemplified in the specification, which aregiven by way of example or illustration and not of limitation. Allquestions concerning scope of the disclosure are to be answered byreference to the appended claims.

1. A photonic crystal biosensor with an integrated fluid containmentstructure, the biosensor adapted for measurement of a sample,comprising: a fluid containment structure having a cavity for receivingsaid sample, said cavity having at least one internal surface; and aphotonic crystal structure comprising a periodic surface gratingstructure formed in the internal surface of said fluid containmentstructure; wherein said fluid containment structure and periodic surfacegrating structure of said photonic crystal sensor comprise an integral,monolithic structure.
 2. The biosensor of claim 1, wherein saidintegral, monolithic structure comprises an integral polymer structurecomprising an optically clear substrate layer, a cured polymer layer,and a relatively high index of refraction material deposited on thecured polymer layer.
 3. The biosensor of claim 1, wherein said cavitycomprises a sample well, and wherein the integral monolithic structurefurther comprises a port for receiving the sample, a channel having asurface, the channel providing a fluid path for connecting the port tothe fluid containment structure, and a second photonic crystal structurecomprising a periodic surface grating structure formed in a surface ofthe channel.
 4. The biosensor of claim 1 wherein said periodic surfacegrating structure comprises a nanosized structure, and wherein saidcavity of said fluid containment structure comprises a micron-sizedfeature.
 5. The biosensor of claim 4 wherein said periodic surfacegrating structure is formed on a bottom surface of said cavity andwherein said periodic surface grating structure extends from a firstside of said cavity to a second side of said cavity.
 6. The biosensor ofclaim 1, wherein the fluid containment structure comprises amicrofluidic flow channel, with the sample flowing through the flowchannel over the photonic crystal structure.
 7. The biosensor of claim1, wherein the fluid containment structure comprises a microwell.
 8. Thebiosensor of claim 1 further comprising a cover layer enclosing saidfluid containment structure.
 9. The biosensor of claim 8, wherein saidcover layer has an inlet hole and an outlet hole for conducting thesample into and out of the fluid containment structure.
 10. Thebiosensor of claim 1 wherein the integral, monolithic structure furthercomprises a polymer substrate supporting said fluid containmentstructure and said photonic crystal structure.
 11. The biosensor ofclaim 1 further comprising a target material bound to the periodicsurface grating structure of the photonic crystal structure.
 12. Thebiosensor of claim 11, wherein said target material is selected from thegroup of target materials consisting of proteins, peptides, DNAmolecules, RNA molecules, oligonucleotides, lipids, carbohydrates,polysaccharides; glycoproteins, lipoproteins, sugars, cells, bacteria,virus, and candidate molecules.
 13. A biosensor comprising: an integral,monolithic structure having a port, a plurality of sample wellsconnected to the port, and a plurality of flow channels connecting theport to the sample wells, and a plurality of photonic crystal sensorsintegrally formed in the structure, wherein the sample wells andphotonic crystal sensors are comprised of a structurally continuousmaterial, each of the sensors positioned in a flow channel connectingthe port to the sample wells.
 14. The biosensor of claim 13, whereineach of the flow channels have substantially the same path lengthbetween the inlet port and the sample wells.
 15. The biosensor of claim13, wherein the plurality of photonic crystal sensors are spatiallyarranged in an aligned condition.
 16. The biosensor of claim 13, whereinthe biosensor comprises an array of N×M sample wells, where N representsan integer number of rows of sample wells and M represents an integernumber of columns of wells, wherein the biosensor includes an inlet portfor each of the M columns of wells, and wherein each of the inlet portsare connected to each of the N wells in each column of wells by a flowchannel, each flow channel in the biosensor incorporating a photoniccrystal sensor.
 17. The biosensor of claim 16, wherein each of thecolumns of wells are in a substantially aligned condition and whereinthe flow channels are constructed such that the photonic crystal sensorsin the flow channels for each column of wells are also in asubstantially aligned condition.
 18. The biosensor of claim 13, whereineach of the sample wells includes a photonic crystal sensor positionedwithin each of the sample wells.